This invention relates to 4-(4-hydroxystyryl) pyridine-containing compounds and the use of 4-(4-hydroxystyryl) pyridine-containing compounds as peroxidase enzyme substrates in a variety of applications such as catalyzed reporter deposition.
Peroxidase, because of its high turnover rate, good stability, and availability is widely used in enzyme-based analytical methods. For example, horseradish peroxidase (HIW) (EC 1.11.1.7) catalyzes the oxidation of a large variety of hydrogen-donating substrates with hydrogen peroxide. HRP is also one of the preferred enzymes for use in catalyzed reporter deposition.
Catalyzed reporter deposition (CARD) is a novel method of signal amplification which constitutes the subject matter of U.S. Pat. Nos. 5,731,158, 5,583,001 and 5,196,306. It is also discussed in Bobrow et al., Journal of Immunological Methods, 125: 279-285 (1989) and in Bobrow et al., Journal of Immunological Methods, 137:103-112 (1991).
The CARD method utilizes an analyte-dependent enzyme activation system (xe2x80x9cADEASxe2x80x9d) to catalyze the deposition of reporter or hapten groups (labels) onto the solid phase of an assay platform. These enzymatically deposited labels are detected directly or indirectly, resulting in signal amplification and improved detection limits. In a preferred embodiment, HRP is the enzyme.
HRP reacts with a conjugate consisting of a labeled compound incorporated into a peroxidase substrate. When the enzyme and the compound react, a reactive intermediate is formed which deposits covalently wherever receptor for the activated reactive intermediate is immobilized. Examples of such compounds which have been described include substituted phenols such as biotinyl-tyramide, fluorescein tyramide and p-hydroxycinnamoyl-containing substrates disclosed in U.S. Pat. No. 5,863,748.
For analytical use, peroxidase substrates have been used to generate products which become colored, fluorescent or chemiluminescent. These products either remain soluble or become insoluble and precipitate on the surface. The CARD method differs in that the reactive intermediates become covalently bound to the surface.
Styryl-pyridinium compounds are well known for use as, for example, electrochromic membrane probes, Loew, Journal of Biochemical and Biophysical Methods, 6 (1982) 243-260; Loew and Simpson, Biophys. J, Vol. 34, June 1981, 353-365; Loew et al., Biochemistry, Vol. 17, No. 19, 1978, 4065-4071; and Loew et al., Nature, Vol. 281, Oct. 11, 1979, 497-499. The use of styryl-pyridinium compounds as probes of membrane potential is specifically set forth in these publications. However, none of these publications teach the use of styryl-pyridinium compounds as substrates.
Typically, fluorescent compounds are used as labels for CARD and other methods. The greater the intensity of fluorescence and the greater the Stokes shift (large spread between excitation and emission maxima) of a fluorescent compound, the lower the detectable limit of the chosen material.
Accordingly, it would be advantageous and desirable to have a labeled compounds which have a high degree of reactivity, a very large Stokes shift, and which also are highly fluorescent when dry, as substrates for use in enzyme assays such as CARD assays.
The present invention relates to labeled 4-(4-hydroxystyryl) pyridine-containing compounds incorporated into peroxidase-based substrates and the use of these substrates in assays. The present invention also relates to 4-(4-hydroxystyryl) pyridine-containing compounds having the structure: 
wherein R1, R2, R3, and R4 are independently H or xe2x80x94Xxe2x80x94L, R5 is an electron pair or xe2x80x94Xxe2x80x94L, X is a linker group capable of linking L to a 4-(4-hydroxystyryl) pyridine moiety, L is a detectable label, A is N or N+, and wherein when R1, R2, R3, and R4 are H, R5 is xe2x80x94Xxe2x80x94L and A is N+ and wherein when any of R1-R4 is xe2x80x94Xxe2x80x94L, A is N or a member of a specific binding pair. The compound itself is fluorescent and may serve as a label. If L is chosen as an appropriate label, fluorescence amplification may be achieved without the use of CARD.
Also disclosed are 4-(4-hydroxystyryl) pyridine-containing compounds incorporated into peroxidase-based substrates and the use of these substrates in assays. The compounds having the structure: 
wherein R1, R2, R3, and R4 are H or Z; R5 is an electron pair or Z, Z is a linear, a branched alkyl, a substituted alkyl, heteroalkyl or substituted heteroalkyl wherein the heteroatom is selected from the group consisting of N, O, or S; A is N or N+ and wherein when R1, R2, R3, and R4 are H, R5 is Z and A is N+ and wherein when any of R1-R4 is Z, A is N.
Also disclosed herein is the use of 4-(4-hydroxystyryl) pyridine-containing compounds in assays for detecting or quantitating the presence or absence of an analyte in a sample which may or may not include the use of CARD to amplify the reporter signal.